Synthesis of steroids



2,914,543 Patented Nov. 24, 1959 j Fire 2 9 4,5 SYNTHESIS or STEROIDS Josef Fried, New Brunswick, and Richard W. Thomas, Somerville, NJ assignors to Olin Mathieson Chemical Corporation, New York, N.Y., a corporation of Virginia No Drawing. Original application March 1, 1957, Serial No. 643,253. Divided and this application January 29, 1959, Serial No. 793,659

4 Claims. (Cl. 260397.3)

This application is a division of our parent application, Serial No. 643,253, filed March 1, 1957.

This invention relates to and .has for its objects the provision of: (1) a microbiological process for preparing 12-oxygenated steroids; and (2) certain new 6,12-oxygenated steroids of the 9(l1)-dehydroprogesterone series.

Prior to this invention, there was no known microbiological method for introducing oxygen into the 12-position of a steroid of the pregnane series (e.g., progesterone and hydroxylated progesterones). We have discovered, however, that if a 9(1l)-dehydro steroid is employed as a substrate, the allylic 12-position becomes vulnerable to the oxidative attack of certain microorganisms; and thereby a 12-hydroxylated steroid can be formed. The microorganisms which will elfect this conversion are those which are known to ll-hydroxylate steroids saturated in the C-ring and unsubstituted in the ll-position (e.g.,

progesterone). Examples of such organisms include Co'lletotrichum phomoides, Thamnidium elegans, Aspergillus nidulans, Tricothecium roseum, Colletotrichum= pisi,

Coniothyrium helleborine, Czmninghamella blakesleeana,

Curvularia lunata, Aspergillus niger, Syncephalastrum V racemosum, Neurospora sitophila, Rhicopus nigricans.

Suitable substrates utilizable in the process of this in vention include steroids of the A -pregnadiene-3,20- dione series, as exemplified by: 9(11)-dehydroprogester one [A -pregnadiene-F:,ZO-dione] A -pre'gnadiene-l7a-ol-3,20-dione; A -pregnadiene-2l-ol-3,20-dione (and the 21-esters thereof, such as the 21'-acetate); A -pregnadiene-l7a,2l-diol-3,20-dione (and the 21-esters thereof); A -pregnatriene-3,20-dione; M pregnatriene 17cc ol 3,20 dione; A -pregnatriene-17a,21-diol-3,20-dione (and the 21-esters thereof); A -pregnatriene-2l-ol-3,20-dione (and the 2l-esters thereof; and A -pregnatriene-3,ZO-dione; also steroids of the A -androstadiene-ii-one series, as ex emplified by: A -androstadiene-3,I'Z-dione; A androstadiene-l7fi-ol-3-one; and 17ot-methyl A -androstadiene-l7fl-ol-3-oue.

The compounds of this invention include GBJZu-dihydroxy-9(ll)-dehydroprogesterone, the diesters thereof and 6,12diketo-9(11)-dehydroprogesterone; and they I may be represented by the general formula:

wherein R is hydrogen, R is hydroxy or acyloxy (particularly the acyloxy radical of a hydrocarbon carboxylic acid having less than ten carbon atoms), or together R and R is keto.

To prepare the 65,l2a-dihydroxy-9(1l)-dehydropro gcsterone of this invention, 9(11)-dehydroprogesterone [A pregnadiene-3,20-dione] is subjected to the action of enzymes of one of the microorganisms listed hereinbefore under aerobic conditions. The hydroxylation can best be eiiected by either including the steroid in an aerobic culture of the microorganism, or by bringing together in an aqueous medium the steroid, air and enzymes of non-proliferating cells of the microorganism.

In general, the conditions of culturing the microor' ganisms for the purpose of this invention are (except for the inclusion of the steroid to be converted) the same as those of culturing various other aerobic fungi for the production of antibiotics, organic acids or vitamins, i.e., the microorganism is aerobically grown in contact with (in or on) a suitable fermentation medium. A suitable medium essentially comprises a source of nitrogenous factors and a source of carbon and energy. The latter may be a carbohydrate (such as sucrose, molasses, glucose, maltose, starch or dextrin), a higher fatty acid, a fat and/or the steroid itself. Preferably, however, the medium includes an assimilable source of carbon and energy in addition to the steroid. The sources of nitrogenous factors may be organic (e.g.,soybean meal, corn steep liquor, meat extract and/or distillers solubles) or synthetic (e.g., composed of simple, synthesizable organic and inorganic compounds, such as ammonium salts, alkali nitrates, amino acids or urea).

An adequate sterile-air supply should be maintained during fermentation, for example, by the conventional methods of exposing a large surface of the medium to air or by utilizing a submerged aerated culture. The steroid may be added to the culture during the incubation period, or included in the medium prior to sterilization or in- :oculation.

The process yields, inter alia, 6fl,12a-dihydroxy-9(11)- dehydroprogesterone, which in turn can be esterified in the usual manner as by treatment with an acyl halide or acid anhydride to yield the diester derivatives. Although any acylating agent may be used, the preferred compounds are the acyl chlorides or acid anhydrides of hydrocarbon carboxylic acids having less than ten carbon atoms, as exemplified by the lower alkanoic acid anhydrides (e.g., acetic anhydride), monocyclic aryl carbonyl chlorides (e.g., benzoyl chloride), monocyclic aralkanoic acid chlorides (e.g., phenacetyl chloride), the lower alkenoic acid anhydrides and the monocycloalkanecarbonyl halides. 1 V

6B,12a-dihydroxy-9 1 1 -dehydroprogesterone is also useful as an intermediate in the preparation of the 6,12- diketo-9(l1)dehydroprogesterone of this invention. This conversion can be effected by oxidation of 6fi,l2a-dihydroxy-9(ll)-dehydroprogesterone with a. hexavalent chromium compound (e.g., chromic acid).

6,8,12a-dihydroxy-9(11)-dehydroprogesterone and its diesters can also be reduced, as by treatment with zinc in acetic acid, to yield l2othydroxy-9(11)-dehydroprogesterone or its ester, respectively. -As disclosed in the US. application of Josef Fried and Josef E. Herz, Serial No. 600,674, filed July 30, 1956, 12a-hydroxy-'9(11)-dehydroprogesterone and its esters are physiologically active steroids which possess progestational activity.

The compounds of this invention are physiologically active steroids which possess progestational activity. Thus, these new steroids can be administered instead of, and in the same manner as, progesterone in the treatment of habitual abortions. The dosage for such administration. is,.of course, dependent on the relative activity of the steroid. Thus, where the steroid in question has approxithe former should be approximately equal to the employed dosage of the latter.

The following examples are illustrative of the invention (all temperatures being in c'entigrade):

EWPLE 1 6B,12m-dihydr0xy-9(11 -dehydrprogesterone Dextrose g Cornsteep liquor g 6 NH4H2PO4 g 3 Yeast extract g 2.5 CaCO g 2.5 Soybean oil g 2.

Distilled water to one liter pH 7.0; sterilized by autoclaving.

After 69 hours of mechanical shaking at 280 rpm. in a 2-inch radius, a 10% (vol/vol.) transfer is made to 75 similar flasks containing the same sterile medium (A). After 44 hours incubation of the second flask stage, 925 mg.' of 9(l1)-dehydroprogesterone in 37.5 ml. methanolic solution is added (25 mg. per flask). After 5 hours further incubation the contents of the flasks are filtered through a Seitz clarifying pad; flasks and pad are washed. The total volume of filtrate and wash is 4150 ml.

(11) Isolation of 6{3,12a-dihydr0xy-9(]1)-dehydr0pr0- gesterone.-The culture filtrate (4150 ml.) is extracted with five l-liter portions of chloroform and the combined extracts evaporated to dryness in vacuo. The semicrystalline residue (about 1.215 g.) is triturated with acetone and the resulting crystals (about 552 mg.) recrystallized from acetone. Pure 6/3,12m-dihydroxy- 9( 1 1 -dehydroprogesterone [A -pregnadiene-6 3,12a diol-3,20-dione] obtained in'this manner has the following properties: M.P. about 231232; [oc] +l07 (c, 0.49 in CHCl REEL 234: m fia- 11,000); h2.963.00, 5.92, 604,621,

The substance gives a characteristic purple color with concentrated sulfuric acid.

AnalysiszCalculated for C l-1 0 (344.44): C, 73.22; H, 8.19. Found: c, 73.40; H, 7.88.

(c) Isolation of A -pregneize-9BJlfl-oxido-fifiJh -diob 3,20-dioneL-The mother liquors from the above crystallization (two experiments) are combined, dissolved in 5 ml. of chloroform and 45 ml. of benzene and chromatographed on 40 g. of. acid-washed aluminum oxide: Elution with chloroform in benzene (1600 ml.) gives amorphous material, which is followed by a crystalline fraction when the eluant is changed to chloroform benzene 1:1. 1600'ml. of the latter solvent mixture elutes 420 mg. of crude crystals, which after recrystallization from acetone gives a pure product, .A -pregnene-9fi,1lfi oxido-6B,12a-diol-3,20-dione, of the following properties: M.P. about 202-204; [u] +4.6 (0,0.41 in CHCl xggzsv mu (e=.15,s00 A213? 2.95, 3.07, 5.95, 6102,

6.18 p. I g V v Analysis: Calculated'for C H O (360.44): C, 69.97; H, 7.83. FoundrC, 69.48; H, 7.79.

Continued. elution of the column withchloroform (650 'ml.) yields an additional amount of 6,8,12a-dih3 droxy 9(11) dehydroprogesterone (about 240 mg). Chloroform containing 5% acetone (300 ml.-), 10%

acetone (200 ml.) and 25% acetone (100 ml.), respectively, elutes mixed fractions, which are followed by another crystalline band when chloroform-acetone 1:1 (800 ml.) is used as the eluant. The material after crystallization from acetone has the following properties: M.P. about 250-252"; [a] +23 (c, 0.30 in CHCI M3, 234 m (e=12,000); Ami 2.95, 5.87, 605 1.;-

Analysis: Calculated for C H O (344.44): 73.22; H, 8.19. Found: C, 72.77; H, 7.58.

A -pregnene-9/3J1,8-oxido-6fi,l2a-diol-3,20-dione is a new steroid which is useful as a progestational agent. Thus, it can be administered instead of, and in the same manner as, progesterone in the treatment of habitual abortions.

EXAMPLE 2 618,1 2oz-dihydr0xy-9(1 1 )-dehydr0pr0gesterone (b) Isolation of 65,120; dihydroxy9(11)-dehydroprogesierone.The culture filtrate is extracted with three 700-ml. portions of chloroform, the solvent evaporated in vacuo and the resulting residue recrystallized from 95% alcohol. Pure 65,12u-dihydroxy-9(11)-dehydro progesterone results, which 'has the following properties identical with those reported in Example 1: M.P. about 230432";

mast-mt (e=11,000

Analysis: Calculated for C H O (344.44): 0,7322; H, 8.19. Found: C, 73.11; H, 8.32.

EXAlVlPLE 3 6,6,12u-dihydr0xy9 (11 -dehydroprogesterone (a) Fermentati0n.The fermentation conditions are the same as those used in Example 1, except the culture is three weeks old and isthat of Aspergillus nidulans ATCC, 11267. Five flasks are used in the first stage, 39 in the second; the first stage is 48 hours in duration; the second stage lasts 29 hours before and24 hours after steroid addition; the amount of steroid is 488 mg; and the volume of'filtrate and wash is 2000 ml.

([2) Isolation of 613,120: dihydroxy 9(11)-dehydr0- pr0gester0ne.Pure 65,120: dihydroxy-9(1l)-dehydro progesterone is isolated from the culture filtrate by the procedure described in. Example 1 section b, and is identical in every respect With the material obtained in Example 1.

EXAMPLE 4 6;3,12a-dihydr0xy-9(11 )-dehydropr0gester0ne 613,120:-

. diacemte 21:. 232 111,. (e=14,400)'; x53 5.75, 5.86, 5.95, 6.17 t.

- Analysis.Calculated for c m o (428.5): c, 70.07;

H, 7.53. Found: C, 70.29; H, 7.01.

EXAMPLE 5 6,12diket0-9 (1 1 -dehydroprogester0ne To a solution of mg. of.6fl,12m-dihydroxy-9(1 l) dehydroprog's'teroiiein'6 ml. of glacial acetic acid is added a solution of 45 mg. of chromic acid in 9 ml. of

glacial acetic acid. After 30 minutes at room temperature 0.5 ml. of alcohol is added, and the mixture is concentrated in vacuo. Water is added and the mixture is extracted with chloroform. The chloroform extract is washed with water and dilute bicarbonate solution, dried over sodium sulfate and evaporated to dryness in vacuo.

The light yellow crystalline residue is recrystallized from acetone and gives pure 6,12-diketo-9(11)-dehydroprogesterone of the following properties: M.P. about 208-210";

[cd +27 (c, 0.38 in CHCl X33, 235-240 m (e: 19,300), 313 m (2,300) h2g2" KOH in MeOH 253 m (19,000), 387 m (7,600) (immediately); 258 m (9,600), 482 m (9,900) (after 3 hours); kg}? 5.90, 5.95, 6.25

Analysis-Calculated for C H O (342.42): C,

74.09; H, 7.11. Found: C, 73.77; H, 7.64.

EXAMPLE 6 12a-hydroxy-9 (11 -dehydropr0gesterone sulfate and evaporated to dryness in vacuo. The amorphous residue is dissolved in 1.5 ml. of benzene and 2 ml. of hexane and chromatographed on 1 gram of acidwashed alumina. Elution with benzene-hexane 1:1 and with benzene filrnishes amorphous material, which is followed by a crystalline fraction (benzene-chloroform 1:1). Recrystallization from acetone-hexane furnishes pure 12a-hydroxy-9(11)-dehydroprogesterone of the following properties: M.P. about 166-168"; [a] +198 (c, 0.27 in CHCl R212 238 my (=16,000); A231 2.96, 5.90, 6.00, 6.20,

In a similar manner, 6B,12a-dihydroxy-9(11)-dehydroprogesterone 65,12a-diacetate can be reduced to 12a-hydroxy-9 1 1 -dehydroprogesterone IZa-acetate.

The invention may be otherwise variously embodied within the scope of the appended claims.

We claim:

1. A compound selected from the group consisting of 6,12-diketo-9( 11)-dehydroprogesterone; 6fi,l2a-dihydroXy-9 1 1)-dehydroprogesterone; and the 65,12a-diesters thereof with hydrocarbon carboxylic acids having less than ten carbon atoms.

2. 6,3, 12a-dihydroxy-9( 11 -dehydroprogesterone.

3. 6,8,120: dihydroxy 9(11) dehydroprogesterone 6B,12u-diacetate.

4. 6,12-diketo-9( 1 1)-dehydroprogesterone.

No references cited.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No, 2,914,543 November 24, 1959 I Josef Fried et a1 It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

In the grant, lines 1 and 2, and in the heading to the printed specification, line 3, name of the second inventor, for "Richard W. Thomas", each occurrence, read Richard Wn Thoma Signed and sealed this 17th day of May 1960.,

KARL H. AXLINE ROBERT C WATSON Attesting Officer I Commissioner of Patents 

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF 6,12-DIKETO-9(11)-DEHYDROPROGESTERONE; 6B,12A-DIHYDROXY-9(11)-DEHYDROPROGESTERONE; AND THE 6B12A-DIESTERS THEREOF WITH HYDROCARBON CARBOXYLIC ACIDS HAVING LESS THAN TEN CARBON ATOMS.
 4. 6,12-DIKETO-9(11)-DEHYDROPROGESTERONE. 